The present invention is directed to a replication competent lentiviral particle that lacks all or a portion of the matrix (MA) protein. Preferably, the particle is a pseudotyped lentiviral particle that contains an envelope protein that targets endocytic compartments, and the vectors that express the particles.
In recent years considerable effort has been directed at applying in vivo gene therapy techniques. That term describes a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. These methods include, for example, vectors such as viral vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. The different techniques used depend in part upon the gene being transferred and the purpose therefore. Thus, for example, there are situations where only a short-term expression of the gene is desired in contrast to situations where a longer term, even permanent expression of the gene is desired.
Vectors that have been looked at include both DNA viral vectors and RNA viral vectors. For example, DNA vectors include pox vectors such as orthopox or avipox vectors (see, e.g., U.S. Pat. No. 4,656,465), herpes virus vectors, such as herpes simplex I Virus (HSV) vector [Geller, A. I. et al., J. Neurochem. 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed., (Oxford Univ. Press, Oxford, England) (1995); Geller, A. I. et al., Proc. Natl. Acad. Sci., U.S.A. 90:7603 (1993); Adenovirus vectors [Legal Lasalle et al., Sci. 259-988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol., 69:2004 (1995); and Adeno Associated Virus Vectors [Kaplitt, M. G., et al.]; Nat. Genet. 8;148 (1994)]. Retroviral vectors include moloney murine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV) [See, U.S. Pat. No. 5,665,577].
While much attention has been focussed on the use of viral vectors, particularly for in vivo therapy, for example, in somatic cell therapy or direct in vivo applications, the clinical testing, particularly large-scale clinical testing is still in its infancy with these vectors. Thus, it is not surprising that results from many of these early trials produces a mixed clinical picture. Nor is it surprising that improvements in viral vectors can still be made.
For example, a retroviral vector can be used to infect a host cell and have the genetic material integrated into that host cell with high efficiency. One example of such a vector is a modified moloney murine leukemia virus (MMLV), which has had its package sequences deleted to prevent packaging of the entire retrovial genome. However, that retrovirus does not transduce resting cells. Additionally, since many retroviruses typically enter cells via receptors, if the specific receptors are not present on a cell or are not present in large enough numbers, the infection is either not possible or is inefficient. Concerns have also been expressed as a result of outbreaks of wild-type viruses from the recombinant MMLV producing cell lines, i.e., reversions.
Recently, attention has focussed on lentiviral vectors such as those based upon the primate lentiviruses, e.g., human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors can infect quiescent cells in addition to dividing cells. Moreover, by using a pseudotyped vector (i.e., one where an envelope protein from a different species is used), problems encountered with infecting a wide range of cell types can be overcome by selecting a particular envelope protein based upon the cell you want to infect. Moreover, in view of the complex gene splicing patterns see in a lentiviruses such as HIV, multivaliant vectors (i.e., those expressing multiple genes) having a lentiviral core, such as an HIV core, are expected to be more efficient. Despite the advantages that HIV based vectors offer, there is still a concern with the use of HIV vectors in view of the severity of HIV infection. Thus, means for providing additional attenuated forms that are less likely to revert to a wild type virus are desirable.
Ultimately, the use of any of these vectors will require the ability to produce such a vector in large scale. Consequently, the method of attenuation and/or preventing reversion should be one that does not adversely affect the ability to have producer cells express large amounts of these vectors. Moreover, the method of attenuation should also not adversely affect the vector""s ability to infect and be expressed in resting cells. Otherwise, one of the substantial advantages of using a lentiviral vector will be lost.
Replication-competent retroviruses contain a matrix (MA) protein which forms a submembrane layer in the mature virion. HIV-1 MA requires a myristylated N-terminus for membrane binding, and has been believed to be essential both for early and late steps of the virus life cycle.
The MA protein is a cleavage product of the polyprotein, Pr55gag, which cleavage is the precursor for the internal structural proteins of the mature virion (Hunter, 1994). Processing of Pr55gag during virus maturation yields the capsid (CA), nucleocapsid (NC), and p6 proteins, in addition to MA.
Pr55gag is cotranslationally myristylated and targeted to the inner leaflet of the plasma membrane, where virus assembly occurs (Hunter, 1994). The assembling particle buds through the plasma membrane and thereby acquires a lipid membrane enriched in viral envelope (Env) glycoproteins. MA forms a spherical shell directly underneath the lipid membrane of the mature virion. CA forms the characteristic conical core, and NC is complexed with the genomic RNA within the core (Hunter, 1994).
Surprisingly, we have found that a lentivirus for example, HIV, feline immunodeficiency virus (FIV), or visna virus, can transduce both dividing and non-dividing cells in the absence of the entire MA or a portion thereof, if a myristylation anchor is provided.
The lentiviral virion (particle) is expressed by at least one vector containing a nucleic acid sequence encoding the lentiviral pol proteins necessary for reverse transcription and integration, operably linked to a promoter. There is also a nucleic acid sequence encoding the lentiviral gag proteins necessary for forming a viral capsid operably linked to a promoter. Preferably, this gag nucleic acid sequence is on a separate vector than the pol nucleic acid sequence. The gag sequence does not express a functional MA protein. This can be accomplished by inactivating the xe2x80x9cgenexe2x80x9d encoding the MA by additions, substitutions or deletions of the MA coding region. Preferably, this is done by deletion. Preferably, at least 25% of the MA coding region is deleted, more preferably, at least 50% is deleted, still more preferably, at least 60%, even more preferably at least 75%, still more preferably, at least 90%, yet more preferably at least 95% and most preferably the entire coding region is deleted.
A myristylation anchor (sequence) is required. Preferably, the myristylation sequence is a heterologous (i.e., non-lentiviral) sequence.
The vector(s) do not contain nucleotides from the lentiviral genome that package lentiviral RNA, referred to as the lentiviral packaging sequence. In HIV this region corresponds to the region between the 5xe2x80x2 major splice donor and the gag gene initiation codon (nucleotides 301-319).
The vector(s) preferably do not contain a nucleic acid sequence from the lentiviral genome that expresses an envelope protein. Preferably, a separate vector contains a nucleic acid sequence encoding an envelope protein operably linked to a promoter. This env vector also does not contain a lentiviral packaging sequence. In one embodiment the env nucleic acid sequence encodes a lentiviral envelope protein. However, the env sequence is altered from the wild type sequence so that it encodes a truncated cytoplasmic tail. Preferably, 50% of the cytoplasmic tail is missing. More preferably, at least 75% is deleted, still more preferably at least 90% is deleted, even more preferably, at least 95% is deleted. Most preferably, the entire cytoplasmic tail is deleted.
In a more preferred embodiment, the env sequence encodes an envelope protein from a different virus such as an envelope protein that targets an endocytic compartment such as that of the influenza virus, VSV-G, alpha viruses (Semliki forest, Sindbis virus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses (tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicular stomatitis virus, rabies virus), and orthomyxoviruses (influenza virus).
The preferred lentivirus is a primate lentivirus [U.S. Pat. No. 5,665,577] or a feline immunodeficiency virus (FIV) [Poeschla, E. M., et al., Nat. Medicine 4:354-357 (1998)] The pol/gag nucleic acid segment(s) and the env nucleic acid segment will when expressed produce an empty lentiviral particle. By deleting the MA coding region, the possibility of a reversion to a wild type virus has been reduced.
A desired heterologous nucleic acid segment can be inserted into the empty lentiviral particle by a vector containing a nucleic acid segment of interest and a lentiviral packaging sequence necessary to package lentiviral RNA into the lentiviral particles. Preferably, the vector contains a 5xe2x80x2 and 3xe2x80x2 lentiviral LTR with the desired nucleic acid segment inserted between them. The nucleic acid segment preferably encodes a protein.
These vectors can be used to express large amounts of viral particles. The particles can be used in a variety of areas. For example, they can be used to generate an immune reaction, to transform a cell with a heterologous nucleic acid sequence and/or to deliver a nucleic acid sequence to a desired host cell.
Pseudotyped lentiviral particles such as HIV-1 particles which lacked the globular head of MA remained fully infectious for macrophages, indicating that MA is dispensable for nuclear import even in terminally differentiated cells.